Process for the digestion of cellulosic material by enzymatic action of trametes suaveolens



Oct. 15, 1968 w, D YERKES JR 3,406,089

PROCESS FOR THE DIGESTION OF CELLULOSIC MATERIAL BY ENZYMATIC ACTION OF TRAMETES SUAVEOLENS Filed Oct. 2, 1964 United States x ABSTRACT OF THE DISCLOSURE A procedure for digesting cellulosic material which procedure is characterized by the enzymatic action of cellulase of Trametes suaveolens derivation. The cellulase in its digestion of the cellulose effects an initial breakdown commonly attributed to the C enzyme and a further attack on linear cellulosic molecules commonly attributed to the C enzyme, both the C and C enzyme being clearly present in, the cellulase of Trametes suaveolens derivation. Additionally, pentosanase activity is demonstrated in the process of application of the cellulase of Trametes suaveolens derivation. The procedure is specifically useful in: the cleaning of equipment by removal of cellulosic materials including the cleaning of papermaking felt s, and in modification of the properties of papermaking pulps.

My present invention relates to the treatment of water insoluble cellulosic materials for the purpose of digesting at least a portion of such materials to water soluble materials; more particularly, this invention is concerned with the provision and use of a novel cellulase for causing cellulosic breakdowns.

In connection with wood degradation in nature, two typesof rot fungi have been commonly described in the literature. In a first one of these, termed brown hot fungi, the fungal action leaves wood or logs in the forest in a brownish appearing condition; this has'been said in the literature to be because the brown rot fungi have attacked the cellulose, to some degree, preferentially to the lignin and removed the cellulose. In contrast, the white rot types of fungi have been considered to attack-lignin primarily, leavingcellulose and resulting in a whitish appearing wood or'logs. Whether the reasoning of the art is correct in theexplanation of the results it is, nevertheless, true that some rot fungi do leave the wood brownish and others leave it whitish.

I have quite unexpectedly found that a type of fungus known in the art as of the white rot type, and which might normally be expected to show a preferential attack on lignin, yields a cellulase which demonstrates a superior activity in its attack on cellulose. My cellulase not only attacks the natural cellulose but is effective to degrade cellulose to glucose; therefore, the cellulase contains apparently both the known C and C types of enzyme. It probably additionally includes the C type of enzyme. In any event the initial cellulose breakdown attributed to the C enzyme and the attack on linear cellulose molecules attributed to the C enzyme is clearly present with my cellulase. Additionally, the new cellulase breaks the pentosans into five carbon sugars and therefore demonstrates a pentosanase activity.

While specific reference has been made hereinbefore to' the attack of this white rot on cellulose, it also does attack lignin to some extent. The utility of the white rot fungus as the source of a material for an attack on lignin is not, however, of particular pertinency in the present "atent O on the treatment of cellulosic fines in crepe wadding pits.

Also, it has considerable capacity for the removal of cellulose from papermaking felts, the type of felts commonly used in papermaking drying operations wherein a felt presses the wet sheet and tends to become loaded with fibrous cellulosic materials. Curiously, however,

while the cellulase of this application is about as effective on carboxymethyl cellulose as are the commercial cellulolytic enzymes, the enzymatic activity of the cellulase of the present application is much more effective on cellulose itself in various forms.

A particularly important consideration in connection with this invention is that the cellulose splitting cellulase is of a high order of activity and studies of the growth variables of the fungus indicate that the potency of the enzymes of the cellulase produced can be increased to a level higher than any other known cellulolytic enzyme. Particularly is this true of the action on celluloseand hemi-cellulose. Thus, my new cellulase is effective not only on cellulosic materials derived from wood by chemical processes such as the sulfite, sulfate and semi-chemical pulps, but also on mechanical pulps including ground wood as well as wood itself.

In essence, then, a primary object of this invention is the provision of a process for digesting cellulosic material and which process is characterized by the enzymatic action of a novel cellulase of particular derivation.

The source of my new cellulase is the fungus Trametes szlaveolens. Trametes suaveolens in nature is characterized as a white heart rotter of Salix or willow and of the poplar also. It grows profusely under laboratory conditions in both still and shaken culture and is very stable in that there is no sectoring or saltation observed. It is easily identified in its culture by its sweet, spicy odor and white floccose growth on solid media. The enzymatic action of the cellulase of the Trametes suaveolens has been found and its effectiveness demonstrated only after the screening of a very great number of fungi.

For the preparation of the cellulase I have-found that a plurality of nutrients are effective. But presently it is much preferred that a combination of ground fir wood and defatted cotton seed meal in aqueous suspension be employed as the nutrient. The cotton seed meal functions to provide the necessary salts, proteins and vitamins for the fungus growth. Such cotton seed meal, though not soluble in aqueous media, disperses readily. During the growth of the fungus air is supplied to the system by bubbling through the suspension, or the suspension is agitated to introduce air. A growth period of 14 days is satisfactory for production of the cellulase containing the enzymes. At the end of this growth period the suspension appears as a light brown clotted mass of firm texture and demonstrating a sweet, spicy smell. This material is then triturated in a Waring Blendor and filtered through diatomaceous earth or any other suitable filter media. The cellulase is contained in the filtrate which is then freed of water, preferably by freeze drying to produce a powder. Freeze drying has the advantage of diminishing the denaturing of the enzymes. The freeze dried product is a very crystalline appearing material considerably proteinaceous in character and which includes the enzymes. The dry cellulase may be reconstituted with water for use. Commonly, 1% by weight of crystals per volume of water, for example, one gram per milliliters of water is satisfactory for use in digestion procedures. The

first successful enrichments of enzymatic activity of the cellulase resulted from the substitution of more complex carbohydrates for glucose in the growing media. Optimum enzyme production as noted has been reached with a combination of mineral nutrient, cotton seed meal, and ground fir substrate. The addition of glucose to such a system, in fact, reduces the yield of potency of the enzyme and, in this respect, is similar to other cellulase producing fungi.

I have found that, as the fineness of the groundwood employed as the carbohydrate source increases, the more effective is the cellulase produced. Fir wood flour extracted with hot water produced a cellulase more effective than fir wood flour alone and more potent than cold water extracted fir flour or hot alcohol extracted fir flour. The substitution of cotton seed protein for neopeptone as the protein source greatly enhances the cellulase production.

In specific application the Trametes suaveolens fungus is grown in an aqueous suspension media containing? ground fir wood of about 200 mesh size and defatted cotton seed meal which disperses readily in the water. The temperature of the medium is maintained at about 25 C. The pH is between about 4.5 and 4.9. During growth air is bubbled into the system over a period of about 14 days. The proportions of the groundwood, cotton seed meal and water may vary widely; a useful combination has been found to be 10 parts by weight of the groundwood to 2 parts by weight of the cotton seed meal and 1000 parts by weight of water. The medium should be sterilized before inoculation.

At the conclusion of the growth period the system is triturated and filtered through diatomaceous earth, the filtrate containing the desired cellulase. The filtrate is then freeze dried; the drying temperature preferably is low-as low as 40 C. below zero. Drying may be carried out, however, at temperatures up to almost +40 C. Freeze drying prevents denaturing of the enzyme. The powder may be stored for long periods of time in a dry atmosphere without deterioration.

The material thus produced is relatively impure but quite useful. By dissolving the impure powder and subjecting the solution to passage through cross-linked dextran gel or the like, to accomplish a molecular sieving, very pure and more effective material on a weight basis may be secured. For most purposes purification is not necessary.

Example 1 In the usage of the enzyme produced as described (without purification) and in specific application, the following procedure was practiced: on 200 milligrams of unbeaten southern pine bleached kraft pulp (oven dry) having a Canadian freeness of about 700 the enzyme was provided as a 1% aqueous solution; this solution was buffered to pH with acetic acid and. sodium acetate. Fifteen ml. of the media were employed to thoroughly wet the pulp. These tubes were mounted on a rotator and the material subjected to slow agitation for 48 hours at a temperature of about 70 F., the speed of rotation being 5 r.p.m. After 48 hours the slurry of fibers was filtered and the fiber washed and oven dried at 40 C. The slurry re sulted from the digestion procedure, the initial material having been at high consistency and in effect simply a wet pad. In fact, the freeness of such. fibrous material is reduced to about 300. The dried fiber was then weighed and the weight loss calculated. The weight loss caused by the cellulase of the invention was about 51%. Comparative tests with two commercially available cellulolytic enzymes each derived from cultures of Aspergillus niger, under the same conditions, resulted in only about a 16% weight loss for a first of the enzymes and a 14% loss for the second commercial enzyme. The fibers resulting from the digestion utilizing the cellulase of the invention appeared under the microscope largely as fiber fragments.

The filtrates in each instance were examined by measuring the amount of glucose equivalent by the Somogi-Nelson copper arseno-molybdate test for sugars. The milligrams of reducing sugars were 97 for the cellulase of this invention, termed herein Ts, 42 for the first of the commercial enzymes, and 34 for thesecond commercial cellulase. The action of the enzymes in similar tests has also shown that pentosans of the'pulpare reduced by the enzymatic action from an average of about 9% to about 5%.

An extensive period of tests has indicated that there is continued digestion of the cellulose by the new cellulase to a period of at least 200 hours, after which time there appears to be very little activity. Varying the concentration of the solution materially influences the rate of digestion of the cellulose. A 1% solution of enzyme is quite efiective. The effectiveness is materially decreased when the concentration is reduced below 0.1%. Below this latter concentration the activity is markedly decreased.

Example 2 Example 1 was repeated at 1% concentration except that the time of digestion was varied from hour to 30 hours and the weight loss at each time interval was determined. Table 1 lists the results.

TABLE 1 Weight loss percent Time (hours): (oven dry basis) It should be noted that even in the hour period there were evidences of glucose and arabinose in the medium, clearly indicating the potency of the enzymatic system.

Example 3 Example 1 was repeated except that the time of digestion was maintained constant and the concentration of the cellulase was varied; the weight loss was determined at each concentration and the data is listed in Table 2.

TABLE 2 Weight loss percent Concentration percent: (oven dry basis) A commercial papermaking felt material designated as Huyck 4D, a product of Huyck Corporation, and loaded with fines from usage in a papermaking operation involving kraft (sulfate) paper production, when subjected to the action of the cellulase of Example 1, results in degradation of the fines to water soluble products. A concentration of 0.1% is effective on such fines. Cellulosic fines present digest in hours and the porosity of the felt materially improves. The felt itself is unaffected. Similar results are obtained in the treatment of other commercial felt materials.

The action of the cellulase on cellulose fibers is illustrated in the accompanying drawings wherein:

FIG. 1 is an illustration based on a photomicrograph of bleached kraft fibers prior to treatment in accordance with the invention; and

FIG. 2 is an illustration also based on a photomicrograph of similar fibers after treatment with the cellulase of the invention in the absence of mechanical action, debris from the fibers which had appeared in the photomicrograph being eliminated for sake of clarity.

As will be noted from FIG. 1, the fibers designated at 1 and shown at about 240 magnification are long, intact, and tend to twist and curl. The fibers designated at 2 generally in FIG. 2 are relatively short, displayed frayed ends as a 3 and, in some instances as shown at 4, the fiber is well digested but not yet completely broken through. Such treatment is relatively extreme and on a weight basis is representative of about a 36% weight loss.

For purpose of fines removal from equipment including pits, felts and the like, such extensive treatment is desirable. For use in connection with the lessening of refining or beating time much less drastic treatment is desired and may be accomplished generally at 0.1% to 1% concentration with only one to two hours treatment. Further mechanical action of the refining or beating process when the enzyme is present during the refining serves to assist in fibrillation and shortening the reaction time. In case of fines removal from pits, felts and the like, the cleaning action is facilitated if the mass of material embodying the fines is scraped or brushed to aid exposure of the finesand vibration of the felt blanket will serve a similar purpose in felt cleaning. Other equipment which is subject to the enzymatic action includes screens in the paper mill, drains and the like. Flushing such equipment at the end of the digestion period removes the active enzymeor the cellulase may be inactivated by treatment with steam or other heat sourcespreferably at a temperature above 140 F.

When employed in beater operation the enzyme may be flushed or washed free from the pulp after the desired digestion has occurred; alternatively, dilution of the pulp for papermaking and subjection of the sheet formed from the pulp to the drying temperature of the paper machine will inactivate the enzyme system permanently, and the formed sheet will not be subject to deterioration.

In general, the pH of the cellulase medium should be between about 4.0 to 7.5 and preferably is about 4.5 to 5.5 for optimum operation. Above about 7.5 the activity of the enzyme decreases significantly. A minimum limit appears to be at about 4 as there is no significant activity at a pH of about 3.8. Additionally, the temperature should be maintained between about C. and 35 C. for most effective digestion; somewhat higher temperatures are permissible at the expense of efiiciency. The quantity of the aqueous medium utilized at any given concentration is not critical but the material should be water wet so that all portions of the material are exposed to the active enzymes.

In addition to the effect on cellulose pulps, the cellulase is effective on other types of cellulosic materials including de-waxed cotton linters and viscose, particularly viscose which has not been stretched in formation. It has been found also that mechanical action on the linters preceding the application of the cellulase or simultaneously therewith aids cellulose breakdown.

An examination of the media after digestion of cellulose as in Example 1 shows that there are present the following constituents in the resultant slurryand which were not present in the starting material:

cellobiose arabinose glucose galactose xylobiose xylose The determination of the presence of these materials has been made by chromatographic analysis. Termination of the action of the cellulose to leave a desired cellulosic water insoluble portion of the cellulose may be effected by, removal of the water insoluble material from the water-solubilized digested material, by excessive dilution of a digesting system preferably followed by washing of the remaining cellulose, by heating the cellulose with the cellulase therein, or by other similar procedural steps.

An important consideration in the utility of the invention is its selectivity in connection with various sizes of fibers. Fines are much more readily attacked than longer fibers and accordingly by control of time of action and concentration one may remove fines from longer fibers, thus improving freeness of pulps.

The cellulose activity is not limited to the cellulosic fibers, fabrics and the like but may also be employed in the clarification of solutions containing cellulosic materials.

What is claimed is:

1. The method of cleaning equipment which is subject in usage to the accumulation of water insoluble cellulosic fines and fibers, which method comprises subjecting the cellulosic fines and fibers to the enzymatic action of cellulase of Trametes suaveolens derivation in aqueous solution at a pH of between about 4 to 7.5 and at a temperature of between about 20 C. and 35 C. to digest the cellulosic containing material to water soluble material, mechanically working the material during digestion, and flushing the digested water soluble material from the equipment.

2. The method of cleaning a papermaking felt of cellulosic water insoluble fines accumulated in the felt which method comprises subjecting the felt with the fines therein to the enzymatic action of cellulase of Trametes suaveolenu derivation in aqueous solution at a pH of between about 4 and 7.5 and a temperature of between about 20 C. and 35 C. to digest the fines to water soluble material and improve felt porosity, vibrating the felt during the digestion action, removing the felt from the said aqueous medium and washing the felt free of said cellulase.

References Cited UNITED STATES PATENTS 2,839,398 6/1958 Stuck 162296 X 3,021,253 2/1962 Opdedbeck 8 X 3,041,246 6/1962 Bolaski et al. 1958 3,310,476 3/1967 Yerkes 195-8 X HOWARD R. CAINE, Primary Examiner. 

